In adults, tissue maintenance and repair depends on a stable population of stem cells that can give rise to both self-renewing and differentiating daughter cells. An understanding of how this process is regulated is of fundamental importance because an excess of differentiation can lead to stem cell depletion and tissue senescence, while a failure to enter the differentiation pathway can lead to an accumulation of proliferating cells and tumor formation. A comparison of male and female germline stem cell (GSC) behavior in Drosophila ovaries and testis, two of the premier model systems for the study of stem cells in their natural environment, reveals sexually dimorphic adaptations of the regulatory mechanisms governing the self- renewal/differentiation decision. Little attention, however, has been paid to how these differences are achieved. Data emerging from the Salz lab supports a novel model in which the female specific RNA binding protein Sex-lethal (Sxl) jointly controls sexual identity and the self-renewal/differentiation decision in the germline. GSCs without Sxl protein fail to successfully execute the self-renewal to differentiation cell fate switch. The failure to differentiate is accompanied by the inappropriate expression of a set of male specific markers, continued proliferation and formation of a tumor. Sxl encodes a ubiquitously expressed female- specific RNA binding protein. In somatic cells it globally regulates all aspects of female-specific development and behavior. Its mode of action in the germline, however, remains poorly understood. The studies in this new proposal address this issue using an integrated experimental approach that combines classical and molecular genetics with RNA/protein biochemistry. In Aim 1 we focus on how Sxl controls germ cell differentiation, beginning with our studies showing that Sxl prompts the exit from the stem cell fate by repressing translation of the conserved stem cell factor nanos. In Aim 2 we focus on how Sxl maintains sexual identity, beginning with preliminary studies showing that Sxl acts through the Jak/Stat signaling pathway to repress male-specific germ cell behavior. The knowledge generated by the studies in this proposal will illuminate the intrinsic regulatory mechanisms that integrate sexual identity and the self- renewal/differentiation decision, and will provide information about why disruptions in this pathway lead to germ cell tumors. Information from model systems such as the Drosophila germline will provide key insights into how stem cells control the self-renewal/differentiation decision in other, less experimentally tractable systems. More generally, the information gained from our animal model studies will illuminate strategies used by RNA regulators to differentially regulate gene expression in time and space.